The overall objectives of this research are to define the gene changes involved in the acquisition of immortalization in mammary epithelial cells which progress to tumors. This objective will be approached by isolating and characterizing unique gene expression in immortalized ductal mammary epithelial outgrowth lines using a differential display PCR method. The RNA expression of isolated genes were be examined in a spectrum of mammary tissues involving normal, hyperplastic and neoplastic states by Northern blots. The functional significance of the isolated genes will be assessed by gene transfer into appropriate mammary epithelial cells. The acquisition of the immortalized phenotype, herein defined as indefinite division potential, is considered to be a rate-limiting step in malignant neoplastic growth culminating in death of the host. Genes or molecular markers related to or specific for immortalization are rare and it is anticipated that this experimental approach will provide new molecular markers and insight into the factors regulating immortalization and neoplastic cell growth.